Submitted to: North Central Avian Disease Conference
Publication Type: Abstract Only
Publication Acceptance Date: 2/22/2005
Publication Date: 3/14/2005
Citation: Pedersen, J.C., Senne, D.A., Suarez, D.L., King, D.J., Kapczynski, D.R., Panigrahy, B. 2005. Modfications of the official real time reverse transcriptase polymerase chain reaction (rrt pcr) assay for the detection of virulent newcastle disease in clinical specimens [abstract]. North Central Avian Disease Conference. p.67.
Technical Abstract: Virulent Newcastle disease (vND) is a serious disease of poultry that has caused severe economic losses in many countries, including the United States. In October of 2002 an outbreak of vND occurred in game fowl in southern California. The disease eventually spread to 21 commercial poultry operations as well as game fowl in Nevada and Arizona. During the outbreak, a Real Time Reverse Transcriptase Polymerase Chain Reaction (RRT PCR) assay was developed and validated to facilitate eradication of vND. Following the outbreak modifications were made to the CalMex (virulent) primers to improve diagnostic sensitivity of the assay. In addition, the Qiagen RNA extraction procedure was compared to the Ambion 96 well magnetic bead procedure for efficiency of RNA isolation and high throughput specimen processing. The volume of swab medium and number of swabs per tube were evaluated as well. The diagnostic sensitivity and specificity of the modified vND virus (vNDV) assay was shown to be 91.26% and 97.5%, respectively. Primer modifications resulted in a significant improvement in diagnostic sensitivity. The CalMex vNDV assay had been shown to have a diagnostic sensitivity and specificity of 85.1% and 99.0% during initial assay validation. The Ambion 96 well magnetic bead extraction system was shown to be more adaptable to high throughput specimen processing and compared favorably to the Qiagen RNeasy RNA extraction kit for the isolation of RNA from cloacal and oropharyngeal swab specimens. The modified vNDV assay has replaced the CalMex assay for the detection of vNDV in the official National Veterinary Services Laboratories RRT PCR protocol used for the surveillance of vNDV currently being conducted by the National Animal Health Laboratory Network